Prions are infectious proteins that cause age-dependent neurodegeneration in humans and animals. The accumulation of an alternatively folded isoform of the prion protein (PrPSc) in the brains of humans and animals is the hallmark of the prion disorders that include Creutzfeldt-Jakob disease (CJD). The prion diseases are invariably fatal and no effective treatment exists. In this application, we propose to continue our studies directed toward devising effective medications for the treatment of CJD. Toward this goal, we want to identify several compounds that either alone or in combination substantially prolong the incubation times in transgenic mice. A model for our proposed research is reflected in our earlier work with bigenic mouse expressing an inducible PrP transgene. These studies showed a substantial prolongation of the incubation time by doxycycline-mediated inhibition of PrPc expression. Toward achieving this goal, we plan to adapt human (Hu) prion protein (PrP) based assays to the High Throughput Screening (HTS) of chemical libraries. Initially, we plan to continue using prion-infected mouse (Mo) neuroblastoma cells designated ScN2a for HTS studies. At the same time, we propose to use uninfected, human (Hu) glioblastoma cells in our HTS search for compounds that decrease HuPrPc levels. In addition, we propose to create cultured cells that propagate either wt Hu or chimeric Hu/Mo CJD prions. These novel cell lines will be used for HTS of chemical libraries. In the proposed studies, both protease-sensitive (s) and resistant (r) PrPSc will be measured in both dividing and non-dividing cells. Potent, non-toxic lead compounds with anti-prion activity will be chemically and computationally optimized by appropriate Structure-Activity-Relationship (SAR) studies. Some computational analyses will focus on the permeability of lead compounds across the bloodbrain- barrier (BBB) and other computational studies on the formation of PrPSc as well as structural models of this protein. Because of problems that plague prion-infected cultured cell assays, we propose to develop additional assays for HTS that include adaptation of an amyloid seeding assay (ASA) using HuPrPSc to seed the polymerization of recombinant (rec) HuPrP as well as a humanized version of a fluorescence polarization (FP) assay that measures the binding of PrP peptides to HuPrPSc. We also propose to use transgenic (Tg) mice expressing either a chimeric Hu/Mo PrP or wt HuPrP that are susceptible to Hu CJD prions. One line of Tg mice exhibits incubation times of ~80 days and these animals should greatly facilitate our studies.